RU2330886C2 - NEW FLUORESCENT PROTEINS Aequorea coerulscens AND METHODS OF APPLICATION THEREOF - Google Patents

NEW FLUORESCENT PROTEINS Aequorea coerulscens AND METHODS OF APPLICATION THEREOF Download PDF

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RU2330886C2
RU2330886C2 RU2004125585/13A RU2004125585A RU2330886C2 RU 2330886 C2 RU2330886 C2 RU 2330886C2 RU 2004125585/13 A RU2004125585/13 A RU 2004125585/13A RU 2004125585 A RU2004125585 A RU 2004125585A RU 2330886 C2 RU2330886 C2 RU 2330886C2
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nucleic acid
cell
polypeptide
protein
fluorescent
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RU2004125585A (en
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Надежда Георгиевна Гурска (RU)
Надежда Георгиевна ГУРСКАЯ
Аркадий Федорович ФРАДКОВ (RU)
Аркадий Федорович ФРАДКОВ
нов Сергей Анатольевич Лукь (RU)
Сергей Анатольевич Лукьянов
Натали Игоревна Пунькова (RU)
Наталия Игоревна ПУНЬКОВА
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Закрытое Акционерное Общество "Евроген Айпи"
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)

Abstract

FIELD: medicine.
SUBSTANCE: invention refers to genetic engineering and biotechnology and can be used for labelling of proteins, cells and organisms. Substitutes added to nucleic acid Aequorea coerulescens, coding colourless GFP-like protein acGFP is considered as means of production of nucleic acid coding fluorescent protein. Expression cassette containing this nucleic acid under regulatory elements control provides in-cell biosynthesis of fluorescent protein. This genetic structure is used for production of transgenic organism and cell producing fluorescent protein, coded by specified nucleic acid. This fluorescent protein is applied in methods for labelling of cells, intracellular structure and protein, as well as for promotor transcriptional activity registration.
EFFECT: invention application ensures widen range of labelling means used in biochemistry, molecular biology and medical diagnostics.
11 cl, 34 dwg, 2 ex

Description

Текст описания приведен в факсимильном виде.

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Claims (11)

1. Выделенная нуклеиновая кислота, кодирующая флуоресцентный полипептид, имеющая нуклеотидную последовательность, по существу, соответствующую открытой рамке считывания аминокислотной последовательности кодируемого флуоресцентного полипептида, где указанная выделенная нуклеиновая кислота выбрана из группы, состоящей из:1. An isolated nucleic acid encoding a fluorescent polypeptide having a nucleotide sequence essentially corresponding to an open reading frame of the amino acid sequence of the encoded fluorescent polypeptide, wherein said isolated nucleic acid is selected from the group consisting of: (a) нуклеиновой кислоты, полученной из нуклеиновой кислоты, которая кодирует белок Aequorea coerulescens acGFP, характеризующийся аминокислотной последовательностью, показанной на фиг. 1, путем введения замен в положения нуклеиновой кислоты Aequorea coerulescens, приводящих к по меньшей мере одной из замен E222G, Y220L, Н148Т или H148S и необязательно к одной или более заменам, выбранным из группы, состоящей из V111, N19D, Т62А, F64L, К101Е, Е115К, N121S, F165L, Е172К, Е172А, Т206А, F221L, Y237I, K238Q в указанной аминокислотной последовательности кодируемого полипептида, и к возникновению у кодируемого полипептида способности к флуоресценции;(a) a nucleic acid derived from a nucleic acid that encodes an Aequorea coerulescens acGFP protein, characterized by the amino acid sequence shown in FIG. 1, by introducing substitutions at the nucleic acid positions of Aequorea coerulescens, resulting in at least one of the substitutions E222G, Y220L, H148T or H148S and optionally one or more substitutions selected from the group consisting of V111, N19D, T62A, F64L, K101E , E115K, N121S, F165L, E172K, E172A, T206A, F221L, Y237I, K238Q in the indicated amino acid sequence of the encoded polypeptide, and the occurrence of fluorescence ability of the encoded polypeptide; (b) нуклеиновой кислоты, кодирующей аминокислотную последовательность, выбранную из группы аминокислотных последовательностей, показанных на фиг. 3, 5, 6, 7, 9, 12, 15, 17, 19 и 21.(b) a nucleic acid encoding an amino acid sequence selected from the group of amino acid sequences shown in FIG. 3, 5, 6, 7, 9, 12, 15, 17, 19 and 21. 2. Выделенная нуклеиновая кислота по п.1, содержащая нуклеотидную последовательность, выбранную из группы нуклеотидных последовательностей, показанных на фиг. 3, 5, 6, 7, 9, 12, 15, 17, 19, 21 и 23, или нуклеотидную последовательность, отличающуюся от указанной вследствие вырожденности генетического кода.2. The isolated nucleic acid according to claim 1, comprising a nucleotide sequence selected from the group of nucleotide sequences shown in FIG. 3, 5, 6, 7, 9, 12, 15, 17, 19, 21 and 23, or a nucleotide sequence that differs from that indicated due to the degeneracy of the genetic code. 3. Кассета экспрессии, содержащая нуклеиновую кислоту по п.1 под контролем регуляторных элементов, необходимых для экспрессии нуклеиновой кислоты в клетке-хозяине, которая будучи интегрированной в геном клетки или при введении в клетку в виде внехромосомного элемента, способна обеспечить экспрессию флуоресцентного полипептида, кодируемого нуклеиновой кислотой по п.1.3. The expression cassette containing the nucleic acid according to claim 1 under the control of regulatory elements necessary for expression of the nucleic acid in the host cell, which, when integrated into the genome of the cell or when introduced into the cell as an extrachromosomal element, is capable of expressing the fluorescent polypeptide encoded nucleic acid according to claim 1. 4. Клетка, продуцирующая флуоресцентный полипептид, кодируемый нуклеиновой кислотой по п.3, содержащая кассету экспрессии по п.3 в виде внехромосомного элемента или элемента, интегрированного в геном этой клетки.4. A cell producing a fluorescent polypeptide encoded by a nucleic acid according to claim 3, containing an expression cassette according to claim 3 in the form of an extrachromosomal element or an element integrated into the genome of this cell. 5. Выделенный полипептид, способный к флуоресценции и кодируемый нуклеиновой кислотой по п.1.5. The selected polypeptide capable of fluorescence and encoded by a nucleic acid according to claim 1. 6. Слитый полипептид, способный к флуоресценции, состоящий из полипептида по п.5 и партнера слияния, представляющего собой направляющий к мишени пептид или сигнальный пептид.6. A fusion polypeptide capable of fluorescence, consisting of the polypeptide according to claim 5 and a fusion partner, which is a peptide or signal peptide that directs the target. 7. Трансгенный организм, содержащий кассету экспрессии по п.3 в виде внехромосомного элемента или интегрированную в геном и способный экспрессировать флуоресцентный полипептид по п.5.7. A transgenic organism containing the expression cassette according to claim 3 as an extrachromosomal element or integrated into the genome and capable of expressing the fluorescent polypeptide according to claim 5. 8. Способ мечения белка, включающий связывание указанного белка с полипептидом по п.5 путем создания нуклеиновой кислоты, кодирующей пептид по п.5, оперативно связанной с нуклеиновой кислотой, кодирующей указанный белок, и получение флуоресцентного слитого белка по п.6.8. A method for labeling a protein, comprising binding said protein to a polypeptide according to claim 5 by creating a nucleic acid encoding a peptide according to claim 5 operably linked to a nucleic acid encoding said protein and obtaining a fluorescent fusion protein according to claim 6. 9. Способ мечения клетки, включающей трансфекцию клетки кассетой по п.3 и продуцирование клеткой флуоресцентного пептида по п.5.9. A method for labeling a cell, comprising transfecting a cell with a cassette according to claim 3 and producing a cell with a fluorescent peptide according to claim 5. 10. Способ мечения внутриклеточной структуры, включающий (а) трансфекцию клетки экспрессионной кассетой, содержащей нуклеиновую кислоту по п.1, слитую с нуклеиновой кислотой, кодирующей сигнал клеточной локализации, специфичный в отношении клеточной структуры, подлежащей мечению, под контролем регуляторных элементов, обеспечивающих продуцирование флуоресцентного полипептида по п.5, слитого с сигналом клеточной локализации, и (b) экспрессию флуоресцентного слитого пептида в клетке.10. A method for labeling an intracellular structure, comprising (a) transfecting a cell with an expression cassette containing the nucleic acid according to claim 1, fused to a nucleic acid encoding a cell localization signal specific for the cell structure to be labeled, under the control of regulatory elements ensuring production a fluorescent polypeptide according to claim 5, fused to a cell localization signal, and (b) expression of a fluorescent fusion peptide in a cell. 11. Способ регистрации транскрипционной активности промотора, включающий (а) продуцирование пептида по п.5 в клетке путем ее трансфекции экспрессирующей кассетой по п.3, содержащей промотор, ответственный в природе за экспрессию изучаемого гена; (b) регистрацию клеточной флуоресценции.11. A method for detecting the transcriptional activity of a promoter, comprising (a) producing a peptide according to claim 5 in a cell by transfecting it with an expression cassette according to claim 3, containing a promoter responsible in nature for the expression of the studied gene; (b) registration of cell fluorescence.
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RU2602452C2 (en) * 2014-05-14 2016-11-20 Федеральное государственное бюджетное учреждение науки Институт биологии гена Российской академии наук Method of substances and their compositions detection with anticancer activity

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WO2010107405A1 (en) * 2009-03-20 2010-09-23 Razumenko Mihail Viktorovich Method for producing peptides which specifically recognize cells of particular types and can be used for therapeutic purposes
RU2602452C2 (en) * 2014-05-14 2016-11-20 Федеральное государственное бюджетное учреждение науки Институт биологии гена Российской академии наук Method of substances and their compositions detection with anticancer activity

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ATE458816T1 (en) 2010-03-15
RU2004125585A (en) 2005-04-20
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DE60331422D1 (en) 2010-04-08
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CA2474108C (en) 2012-06-19
US7667016B2 (en) 2010-02-23
US20060167225A1 (en) 2006-07-27
US20090137033A1 (en) 2009-05-28
IL162568A0 (en) 2005-11-20
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ES2340154T3 (en) 2010-05-31
US7897726B2 (en) 2011-03-01
JP2005526495A (en) 2005-09-08
US7432053B2 (en) 2008-10-07
JP2010046084A (en) 2010-03-04
IL162568A (en) 2010-12-30
US7879988B2 (en) 2011-02-01
US20090148939A1 (en) 2009-06-11
JP4510464B2 (en) 2010-07-21
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